Spectral intensity ratio (sir) analysis for rapid live microbial enumeration

ABSTRACT

Single dye fluorescent staining (with a membrane—associated dye such as FM 1-43 or FM 4 -64) and the combination of differences in both intensity and spectral emission discriminate live from inactivated/dead bacteria and provides for rapid and accurate detection of live bacteria in mixed populations.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.15/542,922, filed Jul. 11, 2017, which is a national stage applicationunder 35 U.S.C. § 371 of International Application No.PCT/IB2016/050094, filed Jan. 9, 2016, which claims the benefit of U.S.Provisional Application No. 62/102,506, filed Jan. 12, 2015 the contentsof each of which is hereby expressly incorporated by reference in itsentirety.

FIELD OF THE INVENTION

Described is a method for rapidly measuring the number of live bacteriain a sample. The method is useful for, e.g., quantitatively validatingthe efficiency of different disinfection procedures that are abundant inmany industries such as pasteurizing, chlorinating, UV, Ozone and forassessing the number of live organisms in food or water for consumption.

BACKGROUND OF THE INVENTION

The level of bacteria in a sample is of great interest to e.g. food andwater intended for human and animal consumption. Many industries such aswater and beverages companies, food industries, pharmaceuticals, etc.,use various disinfection methods for assuring that no bacteria, orminimal amount of bacteria, are left alive in the product after thedisinfection procedures.

Quantification of live microorganisms is typically a specializedlaboratory procedure based upon a bacteria culture growing. Specificconditions for microbial growth on solid and liquid media need to bemaintained over long incubation times at the end of which is determinedthe number of colony forming units (CFU) per unit of volume (e.g.CFU/ml). In the laboratory, the CFU is typically calculated bynormalizing the total number of counted colonies according to the numberof dilutions and the volume of the sample. This methodology requireslaboratory equipment, qualified personnel and long time periods whichmay range from one day to one month. The common procedure is spreading asample on agar plates and incubating them for a period of hours orsometimes days and then counting the number of colonies that grow on theplates. For microorganisms that do not grow on solid medium, andsometimes for anaerobic organisms, the standard is “most estimatedcount” (MEC). (Holms W., J. Gen. Microbiol. 54: 255-260 (1968);Bridgewater L., American Public Health Association, American Water WorksAssociation, and Water Environment Federation, Standard Methods for theExamination of Water and Wastewater. Edited by Eugene W. Rice. 22nd ed.Washington, D.C.: American Public Health Association, 2012). This methoduses tube serial dilution and visualisation of the liquid transparencyfollowed by calculation of starting microbial concentration. Methodsthat require culturing bacteria are, however, slow, expensive, and canbe affected by bacteria clumping, the type of culture media on which theorganism grows, and the presence of dead cells and debris. Additionally,some bacteria will not grow in culture.

It would therefore be advantageous for many industries to have a rapidtool for validating their disinfection protocol by discriminatingbetween live and inactivated bacteria without relying on a culture basedassay.

SUMMARY OF THE INVENTION

The method of the present invention utilizes single dye fluorescentstaining and analysis to create a novel tool for rapid detection of livebacteria. In the method of the present invention, differences in bothintensity and spectral emission are combined to discriminate live frominactivated bacteria.

In one embodiment, the present application provides a method fordifferentiating between live and inactivated bacteria in a treatedsample comprising: staining the sample with a single membrane-associateddye; illuminating the sample with an incident light at λ excitation;measuring, for each bacterial cell (i) the intensity I1 of emitted lightat wavelength λ1; and (ii) the intensity I2 of emitted light atwavelength λ2; calculating a ratio I2/I1; and determining whether thebacterial cell is live or inactivated based on whether the calculatedI2/I1 is larger or smaller than a predetermined threshold. In anotherembodiment, this same process may be conducted for a whole sample ratherthan for each cell individually, measuring whole sample intensity.

In some embodiments, fluorescence analysis, flow cytometry andmicroscopy may be used. Further embodiments, such as (but not limitedto) those involving flow cytometry and microscopy, employ singlebacteria detection and quantification.

The test sample to be analyzed may be a liquid, semi-liquid or drysample. In one embodiment, the sample may be obtained from drinkingwater, a food or a beverage. In a different embodiment, the sample isobtained from a pharmaceutical product, a personal care product, or abody fluid. Body fluids may include, but are not limited to, plasma,saliva, urine, a throat sample, or gastrointestinal fluid. In yetanother embodiment, the sample is obtained from a municipal watersystem, a well, potable water, wastewater, a natural water source,recreational water or a soil. In a different embodiment, the test sampleis from a medical device. Preferably, the medical device is an implant,a patch or a valve.

In some embodiments the membrane-associated dye is a styryl dye. In apreferred embodiment, the membrane-associated dye is FM® 1-43(43N-(3-Triethylammoniumpropyl)-4-(4-(Dibutylamino) Styryl) PyridiniumDibromide) or FM® 4-64(N-(3-Triethylammoniumpropyl)-4-(6-(4-(Diethylamino) Phenyl)Hexatrienyl) Pyridinium Dibromide). In some embodiments, the excitationwave length is between about 360 nm and about 600 nm and the wavelengthsat which I1 and 12 are measured are between about 520 and about 720 nm.In a preferred embodiment, the excitation wavelength is 488 nm and theemission wavelengths at which I1 and I2 are measured are 530 nm and 610nm, respectively. In other embodiments, the excitation wave length isbetween about 360 nm and about 600 nm and the wavelengths at which I1and I2 are measured are between about 600 and about 800 nm. In suchembodiment, the excitation wavelength is in the range of 488 to 570 nmand the emission wavelengths at which I1 and I2 are measured are 670 nmand 780 nm, respectively.

The foregoing general description and the detailed description areexemplary and explanatory and are intended to provide furtherexplanation of the invention as claimed. For detailed understanding ofthe invention, reference is made to the following detailed descriptionof the preferred embodiments, taken in conjunction with the accompanyingdrawings. Other objects, advantages and novel features will be readilyapparent to those skilled in the art from the following detaileddescription of the invention.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a schematic representation of the spectral detection of thestained bacteria and the spectral/intensity discrimination between liveand inactivated bacteria.

FIG. 2 shows fluorescence spectra of: the styryl dye FM® 1-43(43N-(3-Triethylammoniumpropyl) -4-(4-(Dibutylamino) Styryl) PyridiniumDibromide) in stained bacteria: E.coli #8739, 10⁷ cfu/ml in alogarithmic phase, washed and re-suspended in PBS (solid line) and thesame bacteria after inactivation in 85° C. for 15 min (dash line).Bacterial staining was done in 4.9 μM FM® 1-43(43N-(3-Triethylammoniumpropyl)-4-(4-(Dibutylamino) Styryl) PyridiniumDibromide) for 5 min, and washing out the excess dye by centrifugationsteps. Excitation was done at 488nm

FIG. 3 shows normalized fluorescence spectra of: the styryl dye FM® 1-43(43N-(3-Triethylammoniumpropyl) -4-(4-(Dibutylamino) Styryl) PyridiniumDibromide) in PBS (dotted line). E.coli #8739, 10⁷ cfu/ml in alogarithmic phase, washed and re-suspended in PBS (solid line). The samebacteria after inactivation in 85° C. for 5 min (dash line). Bacterialstaining was done in 4.9 microM FM® 1-43(43N-(3-Triethylammoniumpropyl)-4-(4-(Dibutylamino) Styryl) PyridiniumDibromide)for 5 min, washing out the excess dye by centrifugation steps.Excitation was done at 488nm. The double arrows indicate the chosenwavelength for SIR calculation at 530 and 610 nm respectively.

FIG. 4 shows normalized fluorescence spectra of FM® 1-43(43N-(3-Triethylammoniumpropyl) -4-(4-(Dibutylamino) Styryl) PyridiniumDibromide) stained E. coli #8739, 10⁷/ml after different inactivationmethods, as labeled.

FIG. 5 is a Flow-Cytometry dot plots overlay of two bacterial (E.coli#8739, 10⁷ cfu/ml) samples after FM® 1-43(43N-(3-Triethylammoniumpropyl)-4-(4-(Dibutylamino) Styryl) PyridiniumDibromide) staining (left down quadrant) Live bacteria; (right upquadrant) Heated bacteria (pasteurized—85° C., 15 min)), as visualizedwith fluorescent activated cell sorting. The flow cytometry dot plot isgated by light scatter parameters (FSC/SSC) for the bacterialpopulation, the fluorescence filters are 530(30) nm and 610(20)nm.Excitation is by 488 nm laser.

FIGS. 6A-6F shows separated dot plots of E.coli #8739 (10⁷ cfu/ml)samples after FM® 1-43(43N-(3-Triethylammoniumpropyl)-4-(4-(Dibutylamino) Styryl) PyridiniumDibromide) staining: FIG. 6A Live bacteria; FIG. 6B Heated (85° C., 15min), FIG. 6C Chlorinated, FIG. 6D. Ethanol 70%, FIG. 6E. UV-LowPressure, FIG. 6F UV-Medium Pressure. The flow cytometry dot plot isgated by light scatter parameters (FSC/SSC) for the bacterialpopulation, the fluorescence filter are: 530(30) nm and 610(20) nm.Excitation was by 488 nm laser. All inactivated samples (in 6B.-F.)showed 5 to 6 log counting reduction by cfu. The number for X and Y inthe dot-plots represent the mean values of the emission at 530 and 610nm respectively within the marked gate.

FIG. 7 shows normalized fluorescence spectra of FM® 4-64(N-(3-Triethylammoniumpropyl) -4-(6-(4-(Diethylamino) Phenyl)Hexatrienyl) Pyridinium Dibromide) stained E.coli #8739, 10⁷/ml afterdifferent inactivation methods, black solid line—live, dash line—heated,dotted line—chlorinated.

FIGS. 8A-8C is a dot plot overlays of bacterial (E.coli #8739, 10⁷cfu/ml) samples after FM® 4-64(N-(3-Triethylammoniumpropyl)-4-(6-(4-(Diethylamino) Phenyl)Hexatrienyl) Pyridinium Dibromide) staining. FIG. 8A. Live (black dots)vs heated (85° C., 5 min) inactivation (grey dots); FIG. 8B. Live (blackdots) vs chlorine inactivation (grey dots); FIG. 8C. Live (black dots)vs UV-MP inactivation (grey dots). The flow cytometry dot plot is gatedby light scatter parameters (FSC/SSC) for the bacterial population. Thefluorescence filter are: 670(14) nm and 780(60) nm. Excitation was by561 nm laser.

FIGS. 9A-9B is a dot plot overlays of Efaecalis, 10⁷ cfu/ml live, blackdots, and heated (85° C., 15 min), gray dots, stained with (FIG. 9A) FM®1-43 (43N-(3-Triethylammoniumpropyl)-4-(4-(Dibutylamino) Styryl)Pyridinium Dibromide)and (FIG. 9B) with FM® 4-64(N-(3-Triethylammoniumpropyl) -4-(6-(4-(Diethylamino) Phenyl)Hexatrienyl) Pyridinium Dibromide).

FIG. 10 is a graph correlation of bacterial concentration between theflow-cytometry and FM® 1-43(43N-(3-Triethylammoniumpropyl)-4-(4-(Dibutylamino) Styryl) PyridiniumDibromide) calculation and cfu. Including the calculated number and thelog error between the measurements in the small table.

FIG. 11 depicts a graph correlation of bacterial concentration betweenthe flow-cytometry and FM® 4-64(N-(3-Triethylammoniumpropyl)-4-(6-(4-(Diethylamino) Phenyl)Hexatrienyl) Pyridinium Dibromide) calculation and cfu. Including thecalculated number and the log error between the measurements in thesmall table.

FIGS. 12A1-12A3, 12B1-12B3 was produced using microscopy fluorescenceimaging, Excitation.=470 nm, Emission=525(50) nm, of E.coli FM® 1-43(43N-(3-Triethylammoniumpropyl) -4-(4-(Dibutylamino) Styryl) PyridiniumDibromide) stained. [[F]] FIGS. 12A1-A3: Live bacteria. I=35(±5) GL.FIGS. 12B1-B3: Heated inactivated bacteria. I=40(±10) GL. Gray level(GL) analysis performed by Fiji software per object.

FIGS. 13A1-13A3, 13B1-13B3, 13C1-13C3, 13D1-13D3, 13E1-13E3 was producedusing microscopy fluorescence imaging, Excitation.=470 nm,Emission=600(50) nm, of E.coli FM® 1-43(43N-(3-Triethylammoniumpropyl)-4-(4-(Dibutylamino) Styryl) PyridiniumDibromide) stained. FIGS. 13A1-13A3: Live bacteria. I=20-50 GL. FIGS.13B1-13B3: Heated inactivated bacteria. I=128(±20) GL. FIGS. 13C1-13C3:Chlorinated inactivated bacteria. I=135(±20) GL. The sample were treated5 min with 10 mM thiosulfate before microscopic measurements. FIGS.13D1-13D3: Ethanol 70% inactivated bacteria. I=90(±10) GL. FIGS.13E1-13E3: UV-MP inactivated bacteria. I=120(±20) GL. Gray level (GL)analysis performed by Fiji software per object.

FIGS. 14A-14C is a flow-cytometry dot plot of E. coli K12 grown for 5hours followed by (FIG. 14A) 1 hour without antibiotic, (FIG. 14B) 1hour with 700 μg ampicillin, and (FIG. 14C) 1 hour with 1500 μgampicillin.

DETAILED DESCRIPTION

The present invention is based on the finding that that upon stainingbacteria with fluorescent membrane dyes such as styryl dye the emissionfluorescence of live bacteria versus inactivated bacteria are weaker andshifted. Such phenomena might be the result of the interaction of thedyes in the lipophilic membrane environment in the live cells, versusthe inactivated cells where the dyes are inserted to the morehydrophilic environment of the cytoplasm.

As used herein, “live cell” or “live bacteria” means a bacterial cellwhich has the potential to grow and divide. “Dead” and “inactivated” areused interchangeably to refer to dead bacterial cells.

The present invention relates to a method for differentiating betweenlive and inactivated bacteria. The method is performed using spectralintensity ratio analysis of cell membrane dyes. The method is based onthe discovery that, upon excitation of a specimen at a specificwavelength, measurable differences are evident in both the maximumemission peak and emission intensity between live and inactivatedbacteria. Accordingly the ratio of emission intensities at twodesignated wavelengths or spectral intensity ratio (SIR)—I2/I1—can beused as a means of differentiating live bacteria from inactivatedbacteria. The method of the present invention allows accurate and rapiddifferentiation of live from inactivated cells through relying onexcitation/emission based analysis rather than culture based validation,as well as requiring the use of only one dye to successfullydifferentiate. Thus, the method includes steps of: staining the samplewith a single membrane-associated dye; illuminating the sample with anincident light at λ excitation; measuring, for each bacterial cell (i)the intensity I1 of emitted light at wavelength λ1; and (ii) theintensity I2 of emitted light at wavelength λ2; calculating a ratioI2/I1; and determining whether the bacterial cell is live or inactivatedbased on whether the calculated I2/I1 is larger or smaller than apredetermined threshold. In another embodiment, this same process may beconducted for a whole sample rather than for each cell individually. Infurther such embodiments, bulk intensity may be measured to determinewhether the sample contains live or inactivated bacteria.

The system to perform the method of the invention is preferably a devicecapable of excitation of the membrane-associated dye and measuringemission intensity at the prescribed wavelengths λ1 and λ2, such as (butnot limited) to a flow cytometer, fluorescent microscope, or otherinstrument capable of fluorescence analysis.

The emission spectrum profile is measured with a spectral analyser oremission filters. The excitation wavelength is between about 360 nm andabout 600 nm and the wavelengths at which I1 and I2 are measured arebetween about 520 and about 800 nm. In a preferred embodiment, theexcitation wavelength is 488 nm and the emission wavelengths at which I1and I2 are measured are 530 nm and 610 nm, respectively. For FM® 4-64(N-(3-Triethylammoniumpropyl) -4-(6-(4-(Diethylamino) Phenyl)Hexatrienyl) Pyridinium Dibromide) the excitation wavelength could be inbetween 488 to 570 nm and the emission wavelengths at which I1 and I2are measured are 670 nm and 780 nm, respectively

In a preferred embodiment, each bacterial cell is classified as live orinactivated based on the SIR value (I2/I1) relative to the prescribedthreshold.

In some embodiments, the process of determining whether the bacterialcell is live or inactivated based on whether the calculated SIR of thesample is larger or smaller than a predetermined threshold involves anon-viability parameter (NVP) calculated by dividing the SIR of thesample by the SIR of a control of live bacteria. Said NVP is thencompared to a threshold value to determine if the bacteria are live orinactivated. In further embodiments, an NVP of about 1 would indicatelive bacteria while an NVP significantly greater than 1 would indicatedead bacteria. In some embodiments, where the SIR is calculated for thewhole sample, the concentration of the control of live bacteria shouldbe the same as the test sample.

In further embodiments, where the test sample contains a homogeneousbacterial population or heterogeneous bacterial population in a knownratio, a standard curve of SIR values for a control of live bacteria canbe used to establish the predetermined threshold for determining whetherthe bacterial cell is live or inactivated.

Dyes include but are not limited to fluorescent dyes which incorporateinto the lipid bilayer. Examples of fluorescent dyes include styryl dyesand cyanine dyes. Representative styryl dyes include FM® 1-43(43N-(3-Triethylammoniumpropyl)-4-(4-(Dibutylamino) Styryl) PyridiniumDibromide), FM® 1-43FX (Fixable analog of43N-(3-Triethylammoniumpropyl)-4-(4-(Dibutylamino) Styryl) PyridiniumDibromide), FM® 4-64(N-(3-Triethylammoniumpropyl)-4-(6-(4-(Diethylamino) Phenyl)Hexatrienyl) Pyridinium Dibromide) and FM® 4-64FX (fixable analog ofN-(3-Triethylammoniumpropyl)-4-(6-(4-(Diethylamino) Phenyl) Hexatrienyl)Pyridinium Dibromide), FM® 2-10(N-(3-Triethylammoniumpropyl)-4-(4-(Diethylamino)styryl)PyridiniumDibromide) dye. Representative cyanine dyes include Cy2, Cy3, Cy3B,Cy3.5, Cy5, Cy5.5 and Cy7. FM® 1-43 isN-(3-Triethylammoniumpropyl)-4-(4-(Dibutylamino)Styryl)PyridiniumDibromide, purchased from Life Technology (#T-35356), and also sold bySigma as “Synaptogreen” (#S6814). FM® 4-64 isN-(3-Triethylammoniumpropyl)-4-(6-(4-(Diethylamino) Phenyl) Hexatrienyl)Pyridinium Dibromide purchased from Life Technology (#T-13320) or Sigmaas “Synaptored” (#S6689).

More than one dye can be used, but the present method may be performedwith a single dye. The use of a single dye not only simplifies themethod, but reduces variability caused by the presence of two dyes.

The test sample to be analyzed may be a liquid, semi-liquid or drysample. The sample may be obtained from drinking water, a food or abeverage, a pharmaceutical product, a personal care product, or a bodyfluid. Body fluids may include, but are not limited to, plasma, saliva,urine, a throat sample, or gastrointestinal fluid. Test samples may alsobe obtained from a municipal water system, a well, potable water,wastewater, a natural water source, recreational water or a soil. Indifferent embodiments, test samples are obtained from medical devices.Examples of medical devices include, but are not limited to, implants,patches and heart valves.

In some embodiments, the test sample may be analyzed for success orfailure of a bacterial inactivation treatment, such as (but not limitedto) antibiotic or antibacterial treatment, Chlorine inactivation,heating, Ethanol, and UV irradiation by medium pressure. In furtherembodiments, a threshold value can be determined by taking the I2/I1 ofa pre-treatment sample and then compared to the I2/I1 of the test sampleto determine efficacy of the bacterial inactivation treatment.

Bacteria may include, but are not limited to, coliform bacteria,enterobacteria, Salmonella, Listeria, Shigella, Pseudomonas,Staphylococcus or Methanobacterium.

The following examples are set forth as representative of specific andpreferred embodiments of the present invention. These examples are notto be construed as limiting the scope of the invention in any manner. Itshould be understood that many variations and modifications can be madewhile remaining within the spirit and scope of the invention.

Materials and Methods

Dyes: N-(3-Triethylammoniumpropyl)-4-(4-(Dibutylamino) Styryl)Pyridinium Dibromide purchased from Life Technology as FM® 1-43(#T-35356) or Sigma as “Synaptogreen” (#S6814). N-(3-Triethylammoniumpropyl)-4-(6-(4-(Diethylamino) Phenyl) Hexatrienyl)Pyridinium Dibromide purchased from Life Technology as FM® 4-64(#T-13320) or Sigma as “Synaptored” (#S6689).

Bacteria: Unless otherwise stated, bacteria were purchased fromBiological Industries, Beit Haemek, Israel. Dried bacteria was recoveredaccording to ATCC instruction and aliquots were stored in glycerol in−20° C.

E. coli ATCC (#8739) E. coli ATCC (#25922) E. coli O157:H7 (EHEC) E.aerogenes ATCC (#13048) E. cloacae ATCC (#23355) C. freundii ATCC(#8090) K pneumonia ATCC (#13883) E. faecalis ATCC (#19433) E. faecalisATCC (#35550) E. faecium ATCC (#19434) S. aureus ATCC (#25923) B.subtilis ATCC (#6633) Ps. Aeruginosa ATCC (#9027) Ps. Aeruginosa ATCC(#27853)

Bacterial inactivation: Bacteria in phosphate buffered saline (PBS) wereinactivated by one of the following procedures: Pasteurization(Heating)—85° C., 5-15 min; Chlorine (Sodium-hypochlorite) —0.01%, 10min; Ethanol—70%, 10 min; UV-MP irradiation—4-70 mJ, medium pressurelamp (200-300 nm) in Atlantium Tech. LTD; UV-LP irradiation: 4-70 mJ,Low pressure lamp (254 nm) in Atlantium Tech. LTD.

Microscopy: Live or inactivated bacteria was stained with FM®1-43(43N-(3-Triethylammoniumpropyl) -4-(4-(Dibutylamino) Styryl)Pyridinium Dibromide) (4.9 μM) or Synaptored (15 μM) in PBS for 5 min inroom temperature. Using TACounts unique filtration system, the samplewas filtered through a polycarbonate membrane with 0.4 μm cutoff. Thefilter was washed using 1 ml PBS and then imaged using a fluorescencemicroscope (Axio Scope A1, Zeiss, Germany).

Excitation/emission filters based on Cube38 in the Zeiss setup. For FM®1-43 (43N-(3-Triethylammoniumpropyl) -4-(4-(Dibutylamino) Styryl)Pyridinium Dibromide) - Excitation: 470 (±20) nm, Beam Splitter FT495,Emission: BP 525 (±25) nm or 600 (±25) nm. For FM® 4-64(N-(3-Triethylammoniumpropyl) -4-(6-(4-(Diethylamino) Phenyl)Hexatrienyl) Pyridinium Dibromide)-Excitation: 535±25 nm, Emission:590LP.

Quantitative microscopic image analysis was conducted using Fijisoftware. Gray levels (GL) are independent of color and indicate thebrightness of individual pixels, taking into account color sensitivityof the human eye.

Flow Cytometry: Live or inactivated bacteria was stained with FM® 1-43(43N-(3-Triethylammoniumpropyl) -4-(4-(Dibutylamino) Styryl) PyridiniumDibromide) (4.9 μM) or FM® 4-64(N-(3-Triethylammoniumpropyl)-4-(6-(4-(Diethylamino) Phenyl)Hexatrienyl) Pyridinium Dibromide)

(15 μM) in PBS for 5 min in room temperature. Preliminary work showedthat immediate measurements does not require washing steps. The sampleswere measured using fluorescent activated cell sorting (FACS. Here,FACSARIA III, BD, USA, was used). The optical setup for FM® 1-43(43N-(3-Triethylammoniumpropyl)-4-(4-(Dibutylamino) Styryl) PyridiniumDibromide) stained bacteria include light scatter parameter by theprimary 488 nm excitation and FSC/SSC detector and fluorescencemeasurements using band path filters of 530 (±15) nm, 610 (±10) nm. Forthe FM® 4-64 (N-(3-Triethylammoniumpropyl)-4-(6-(4-(Diethylamino)Phenyl) Hexatrienyl) Pyridinium Dibromide) stained bacteria thefluorescence parameters were detected by the 561 nm laser excitation andusing band path filters of 670±7 nm and 780±30 nm. Results wereprocessed using FCS Express 4 (De Novo Software, USA).

Spectroflourimetry: 10⁷/ml Live or inactivated bacteria in PBS werestained with FM® 1-43(43N-(3-Triethylammoniumpropyl)-4-(4-(Dibutylamino) Styryl) PyridiniumDibromide) or FM® 4-64(N-(3-Triethylammoniumpropyl)-4-(6-(4-(Diethylamino) Phenyl)Hexatrienyl) Pyridinium Dibromide) as described. Bacteria werecentrifuged (14,000 rpm, 3 min) and re-suspended in 1 ml PBS. 250 μl ofbacteria solution were transferred to dark 96 well plate (BrandTechScientific). Samples stained with FM® 1-43(43N-(3-Triethylammoniumpropyl)-4-(4-(Dibutylamino) Styryl) PyridiniumDibromide) were excited with 490 nm and fluorescence spectrum wasmeasured between 520 nm and 700 nm (Synergy H1 Multi-Mode Reader,Bio-Tek, Canada). Samples stained with FM® 4-64(N-(3-Triethylammoniumpropyl)-4-(6-(4-(Diethylamino) Phenyl)Hexatrienyl) Pyridinium Dibromide) were excited with 490 nm andfluorescence spectrum was measured between 530 nm and 850 nm (Infinite200 Pro, Tecan, Switzerland).

EXAMPLES

The following examples illustrate the invention, and are not intended tobe limiting.

Example 1—Scanning Fluorescence Results for FM® 1-43(43N-(3-Triethylammoniumpropyl)-4-(4-(Dibutylamino) Styryl) PyridiniumDibromide) Stained Bacteria

The fluorescence spectra of the styryl dye (FM® 1-43(43N-(3-Triethylammoniumpropyl) -4-(4-(Dibutylamino) Styryl) PyridiniumDibromide)) and the stained bacteria in PBS are presented in thefluorescence spectra in FIG. 2 and in its normalized form in FIG. 3 forenhancing the visibility of the spectral changes. It is clearly shownthat the maximum emission peak of the dye is shifted in the livebacteria towards the blue, compare to the inactivated bacteria and tothe free dye. The maximal peaks are: 635 nm—free dye, 595 nm for theinactivated bacteria and 560 nm for the live bacteria. It is also shownthat the intensity of the fluorescence is lower in the live bacteria,with I=3000 at 560 nm, compare to the inactivated bacteria, whereI=40,000 at 595 nm. It is therefore evident that differentiation betweenlive and inactivated bacterial populations can be made using the twoparameters—the spectral shift and intensity. To quantify the phenomenathe ratio between the fluorescence intensities in the two distinctwavelength, which will reflect the maximum effect and be useful forfuture implementation, was calculated. The selected wavelength as markedin FIG. 3 are 530 nm and 610 nm. The Intensity values of thesewavelength are presented in Table 1. One can see that for theinactivated bacteria the fluorescence intensity increased 4 fold in 530nm and 20 fold in 610 nm. This indicates that not only there isincreased fluorescence staining in the dead bacteria but thefluorescence spectra is shifted. The different enhancement in the twowavelengths for the inactivated bacteria compared to the live bacteriaenable us to calculate the SIR and the NVP, and summarize in Table 1.

The same spectral analysis was obtained in different inactivationmethods such as Chlorine inactivation, heating, Ethanol and UVirradiation by medium pressure and low pressure. For the method theviability reduced in 5 to 6 logs as shown by cfu method. The overlay ofthe outcome fluorescence spectra is shown in FIG. 4, and summarized inTable 1. One can notice that in all the method, except for the UV-LP,the NVP>1, which correlate to the non-viability state of the bacteria asdemonstrated in cfu. Treating the bacteria by UV-LP indeed causes theirinactivation (5 logs) however SIR effect didn't occur, this is probablydue to the fact that in UV-LP the irradiation is limited to 254 nm whereonly DNA is damaged and not the protein or the cell membrane. It isknown that UV irradiation can damage DNA, leading to cells that areunable to replicate, but remain able to metabolize for a period of time.

TABLE 1 SIR and NVP calculation from the fluorescence intensities valuesderived from the fluorimeter measurements of E.coli-FM ® 1-43(43N-(3-Triethyl- ammoniumpropyl)-4-(4-(Dibutylamino) Styryl) PyridiniumDibromide) staining* 85°, Chlorine Ethanol UV-MP** UV-LP Wavelength Live15 min 0.05% 70% 30 mJ 30 mJ SIR (I₆₁₀/I₅₃₀) ~1.5 ~7 ~8 4.4 3.7 1Non-Viability ~5 ~5 ~3 ~2 1 Parameter (NVP) *All samples must containsame bacterial concentration for comparable intensity measurements.**Irradiation of the bacteria with medium pressure UV (UV-MP) at therange of 200-300 nm results in reduce viability of at least 5 log in therange of 4 to 70 mJ. Irradiation with low pressure UV (UV-LP) alsoresult in the bacteria inactivation but with no SIR effect.

Example 2—Flow Cytometry results FM® 1-43(43N-(3-Triethylammoniumpropyl)-4-(4-(Dibutylamino) Styryl) PyridiniumDibromide) Stained Bacteria

Although fluorimeter measurements are quantitate, it is a macroscopicmeasurement of the total sample, hence it is dependent on the bacteriaconcentration in the solution. To verify that the phenomena can bedetected also with a single bacteria, Flow-Cytometry measurements of thestained bacterial samples were performed. In order to adjust theFlow-Cytometry to the spectral characterization as shown in thefluorescence spectra of the FM® 1-43 (43N-(3-Triethylammoniumpropyl)-4-(4-(Dibutylamino) Styryl) Pyridinium Dibromide) stained bacteria, the488 nm excitation channel of the flow-cytometry and two fluorescencechannel using 530 (30) nm and 610 (20) nm as the λ1 and λ2 for the SIRcalculation were used.

In FIG. 5 the overlay dot plots of live bacteria, E. coli, vs.inactivated (by heat) bacteria is shown on the fluorescence parameter of610/530 nm. It is clear that the inactivated population has enhancedfluorescence in both wavelength 610 nm and 530 nm, which is moredominant in the 610 nm.

The process described was repeated with the same bacteria for fivedifferent inactivation procedures and their fluorescence results shownin the scanning fluorimeter above and here in the flow cytometryanalysis of the 610/530 nm emission filters. The resulted dot plots forthe different bacterial status flow-cytometry analysis is presented inFIGS. 6A-6F. For each dot plot the gated population represent thebacteria and its mean fluorescence emission for 530 and 610 nm is shownas X and Y respectively. From these values the SIR can be calculated.For example for live bacteria the mean fluorescence intensities are:I₅₃₀=42 and I₆₁₀=31 to yield SIR_(live)=0.73. For the heated inactivatedbacteria the mean fluorescence are: I₅₃₀=457 and I₆₁₀=4045 to yieldSIR_(heated)=8.8. Hence the NVP for the heated bacteria would be 9. Therest of the samples SIR and NVP values are summarized in Table 2. In allcases of inactivated bacteria the SIR and consequently the NVP valuesare greater than 1 except for the UV-LP treated bacteria. Moreover, theconcluded data from the flow-cytometry are in line with the data fromfluorimeter measurements. Although somewhat different, it does show asimilar magnitude and trend.

The UV-LP irradiation (limited to 254 nm) causes inactivation of thebacteria only by DNA damage with no membrane or protein inactivation,likely maintaining the bacterial structure at the molecular level. Thus,UV-LP irradiation inactivation may not result in inactivated bacteriawith different staining of the FM® 1-43(43N-(3-Triethylammoniumpropyl)-4-(4-(Dibutylamino) Styryl) PyridiniumDibromide) when compared to live bacteria. However, UV-LP is not arecommended treatment for bacteria in large water treatment. Indeed, forlarge water treatment UV-MP treatment is recommended by the authoritiessuch as EPA; for UV-MP the calculated NVP was 5.

TABLE 2 SIR and NVP calculation from the fluorescence intensities valuesderived from the Flow-Cytometry measurements of E.coli-FM ® 1-43(43N-(3-Triethyl- ammoniumpropyl)-4-(4-(Dibutylamino) Styryl) PyridiniumDibromide) staining 85°, Chlorine Ethanol UV-MP** UV-LP Wavelength Live15 min 0.05% 70% 30 mJ 30 mJ SIR (610/530) Ratio 0.73 8.8 9.6 6.5 3.50.7 Non-Viability 9 10 7 5 1 Parameter (NVP)

Example 3—FM® 4-64 (N-(3-Triethylammoniumpropyl)-4-(6-(4-(Diethylamino)Phenyl) Hexatrienyl) Pyridinium Dibromide) Stained Bacteria

Another member of the styryl dyes is Synaptored or FM® 4-64(N-(3-Triethylammoniumpropyl) -4-(6-(4-(Diethylamino) Phenyl)Hexatrienyl) Pyridinium Dibromide) which is characterized in longerhydrophobic region and bathochromic emission characterization(atλmax=750 nm) then the FM® 1-43(43N-(3-Triethylammoniumpropyl)-4-(4-(Dibutylamino) Styryl) PyridiniumDibromide).

In FIG. 7 one can observe the resulting normalized fluorescence spectraof live, heated (85°, 15 min) and chlorinated stained E.coli with theFM® 4-64 (N-(3-Triethylammoniumpropyl)-4-(6-(4-(Diethylamino) Phenyl)Hexatrienyl) Pyridinium Dibromide). For the live bacteria the maximumemission peak is shifted to 690 nm, for the inactivated bacteria maximumemission peak are at 740 and 760 nm respectively. From the spectra onecan define two wavelength for calculating SIR, which are at 780 nm and670 nm. These wavelength were used as optical filters in a FlowCytometry set up for the SIR analysis per single bacteria. In FIGS.8A-8C we can see the dot-plots overlay of live, heated, and chlorinatedbacterial population on the 670(14)/780(60) nm emission channels. As canbe seen the inactivated bacterial population is in the low 670/780channels where for the inactivated bacterial population the emission onthe 780 (60) nm channel is increased. For calculating the SIR wasderived from the flow cytometry statistic the mean fluorescenceintensity in both wavelength to calculate the SIR values and the NVP.These values, Table 3, show correlation between the inactivation statesof the bacteria to the NVP value, as for the inactivated bacteria NVP>1.

Comparing the NVP values of E.coli stained with FM® 4-64(N-(3-Triethylammoniumpropyl) -4-(6-(4-(Diethylamino) Phenyl)Hexatrienyl) Pyridinium Dibromide) to FM® 1-43(43N-(3-Triethylammoniumpropyl)-4-(4-(Dibutylamino) Styryl) PyridiniumDibromide) reveals more distinct values for FM® 1-43(43N-(3-Triethylammoniumpropyl)-4-(4-(Dibutylamino) Styryl) PyridiniumDibromide), however both dyes show NVP>1, hence can be used todiscriminate Live/Inactivated bacteria.

TABLE 3 SIR and NVP calculation by Flow-Cytometry analysis at 670(14)/780 (60) nm channels for FM ® 4-64(N-(3-Triethylammoniumpropyl)-4-(6-(4-(Diethylamino) Phenyl)Hexatrienyl) Pyridinium Dibromide) stained E.coli #8793. For the heatedand chlorinated bacteria cfu results show 6 log decreasing. Bacteriastatus SIR calculation NVP calculation Live 3.5 Heated (85°, 15 min) 144 UV-MP 13 3.7 Chlorinated 12 3.4

Example 4—Gram Positive Bacteria

Gram positive bacteria are different in their membrane structure fromGram negative by having thicker peptidoglycan layer on the outer side ofthe cell membrane where in the Gram negative the thin peptidoglycan isinside an outer cell membrane. Since the type of staining presented hereis membrane dependent, it was expected that gram positive bacteria wouldbehave differently from gram negative bacteria. We have observed thatfor gram positive bacteria the phenomena is weaker with the FM® 1-43(43N-(3-Triethylammoniumpropyl)-4-(4-(Dibutylamino) Styryl) PyridiniumDibromide) dye and it is more dominant using the more lypophylic dye,FM® 4-64 (N-(3-Triethylammoniumpropyl)-4-(6-(4-(Diethylamino) Phenyl)Hexatrienyl) Pyridinium Dibromide).

While differences were observed, we were surprised that both grampositive and negative bacteria can examined in this method.

As an example to Gram positive bacteria Enterococcus faecalis bacteria,which is an important water infectious indicator has been stained andanalyzed by fluorimeter analysis (data not shown) and flow cytometryanalysis. In FIG. 9 the flow cytometry analysis of the stainedE.faecalis as live and inactivated by heat are presented, both by FM®1-43 (43N-(3-Triethylammoniumpropyl) -4-(4-(Dibutylamino) Styryl)Pyridinium Dibromide) (FIG. 9A) and FM® 4-64(N-(3-Triethylammoniumpropyl)-4-(6-(4-(Diethylamino) Phenyl)Hexatrienyl) Pyridinium Dibromide) (FIG. 9B) staining. Other means ofinactivation like chlorine and UV_MP show similar dot plots overlay. Itis clear that in both staining there are different SIR for theinactivated compare to the live bacteria, with a stronger effect usingthe FM® 4-64 (N-(3-Triethylammoniumpropyl) -4-(6-(4-(Diethylamino)Phenyl) Hexatrienyl) Pyridinium Dibromide) staining. The SIR and NVPwere calculating by using the flow-cytometry gating statistic data andpresented in Table 4. Both FM® 1-43(43N-(3-Triethylammoniumpropyl)-4-(4-(Dibutylamino) Styryl) PyridiniumDibromide) and FM® 4-64(N-(3-Triethylammoniumpropyl)-4-(6-(4-(Diethylamino) Phenyl)Hexatrienyl) Pyridinium Dibromide) show NVP >1 for the inactivatedbacteria with a higher results for the FM® 4-64(N-(3-Triethylammoniumpropyl)-4-(6-(4-(Diethylamino) Phenyl)Hexatrienyl) Pyridinium Dibromide). Better Live/Inactivateddiscrimination with the more hydrophobic dye is, thus, demonstrated.

TABLE 4 SIR and NVP calculation by Flow-Cytometry analysis forE.faecalis Live and inactivated (heated and chlorine) after FM ® 1-43(43N-(3-Triethyl- ammoniumpropyl)-4-(4-(Dibutylamino) Styryl) PyridiniumDibromide) and FM ® 4-64(N-(3-Triethylammoniumpropyl)-4-(6-(4-(Diethylamino) Phenyl)Hexatrienyl) Pyridinium Dibromide) staining. Inactivated bacteria are5-6 log decreased by cfu. Live Heated Chlorine UV-MP FM ® 1-43(43N-(3-SIR: 6.5 10.3 13 7.1 Triethylammoniumpropyl)- 530(30)/610(20)4-(4-(Dibutylamino) Styryl) NVP 1.6 2 1.1 Pyridinium Dibromide)FM ® 4-64 (N-(3- SIR: 6.7 20 19.7 8.1 Triethylammoniumpropyl)-670(14)/780(60) 4-(6-(4-(Diethylamino NVP 3 2.9 1.2 Phenyl) Hexatrienyl)Pyridinium Dibromide)

Example 5—Expanding the Effect across Several Bacteria

All Gram negative have significant SIR effect for all inactivationmethods in here: UV-MP, Heating and Chlorinated. FM® 1-43(43N-(3-Triethylammoniumpropyl)-4-(4-(Dibutylamino) Styryl) PyridiniumDibromide) is preferred than FM® 4-64(N-(3-Triethylammoniumpropyl)-4-(6-(4-(Diethylamino) Phenyl)Hexatrienyl) Pyridinium Dibromide) for Gram negative.

Gram positive show SIR effect weaker but positive, better in FM® 4-64(N-(3-Triethylammoniumpropyl) -4-(6-(4-(Diethylamino) Phenyl)Hexatrienyl) Pyridinium Dibromide). For UV-MP results are low.

UV-LP—no effect

TABLE 5 NVP values, calculated by Flow-Cytometry analysis of severalGram negative and positive bacteria by FM ® 1-43(43N-(3-Triethylammoniumpropyl)-4-(4- (Dibutylamino) Styryl) PyridiniumDibromide) and FM ® 4-64 (N-(3-Triethylammoniumpropyl)-4-(6-(4-(Diethylamino) Phenyl) Hexatrienyl)Pyridinium Dibromide) staining FM ® 4-64 (N-(3- FM ® 1-43 (43N-(3-Triethylammoniumpropyl)- Triethylammoniumpropyl)- 4-(6-(4-(Diethylamino)4-(4-(Dibutylamino) Styryl) Phenyl) Hexatrienyl) Staining PyridiniumDibromide) Pyridinium Dibromide) Inactivation bacteria Chlorine HeatedUV-MP Chlorine Heated UV-MP Coli- E.coli- 10 9 5 3.4 4 3.7 form #8739E.coli- 9 7 5 #25922 E.coli-O157 ~10 ~10 Nd Entrobacter 10 8 5 4.5 3.72.5 cloacae Entrobacter 12 8 4.5 4.5 3.3 1.7 aerogenes Pseudomonas 5 4.63.2 2 1.8 aeruginosa Gram Enterococcus 2 1.6 1.1 2.9 3 1.2 Positivefaecalis Bacillus 1.5 1.2 1.2 1.5 1.8 subtillis Enterococcus 1.7 1.6 Nd3 3 faecium S.aureus 1.7 1.6 1.1 2.4 3 1.1

Counting bacteria: The double fluorescence analysis and the SIR effectof the bacteria by the FM® 1-43(43N-(3-Triethylammoniumpropyl)-4-(4-(Dibutylamino) Styryl) PyridiniumDibromide) or FM® 4-64(N-(3-Triethylammoniumpropyl)-4-(6-(4-(Diethylamino) Phenyl)Hexatrienyl) Pyridinium Dibromide) staining and flow cytometry analysisenable to discriminate the events representing live bacteria from otherevents representing debris, aggregate and inactivated bacteria asdescribed. These events, corresponding to the NVP of live bacteria, arecounted during the flow-cytometry analysis. The concentration of thesamples can be derived by knowing the flow rate and the time of analysisof each samples, as previously described in the literature. Equation 1translates the event count (g) to bacteria concentration per ml (C):

[C]=g*1000/(flow rate)*time   Equation 1

Where flow rate is in microL/min, and time is in min.

The results are presented for FM® 1-43(43N-(3-Triethylammoniumpropyl)-4-(4-(Dibutylamino) Styryl) PyridiniumDibromide) and FM® 4-64(N-(3-Triethylammoniumpropyl)-4-(6-(4-(Diethylamino) Phenyl)Hexatrienyl) Pyridinium Dibromide) staining in FIG. 10 and respectivelyFIG. 11. There is very high correlation (r²>0.99) between the twomethods on the range of 1000 to 10⁶ bacteria. In the low level of lessthan 1000 the noise may outnumber the signal without any ability todiscriminate noise from bacteria. In all concentrations (between 1000 tomillion) the error is less than 0.5 Log as accepted in microbiologicalcounting.

Example 7—Microscopy Results

Observing live bacteria and inactivated bacteria in the standardemission filter, 525(50) nm, in the fluorescence microscope shows thatthere is little difference in intensity, see FIGS. 12A1-12B3, which isin line with the findings of the present invention. When observing thefluorescence bacteria thru the 600(50) nm emission filter, the change inintensity from 20-50 GL in live bacteria to the range of 90-150 indifferent inactivated bacteria, FIGS. 13A1-13E3, is evident.

Example 8—E. coli K12 with Ampicilin

The disclosed method was further validated in studies using E. coli K12(MG1655, obtained from ATCC). After 5 hours of growth in medium, E. coliwere divided into three groups: (A) grown for one hour with noantibiotics, (B) grown for one hour with 700 micrograms of ampicillin,and (C) grown for one hour with 1500 micrograms of ampicillin.

The bacteria were suspended in PBS and stained with FM® 1-43(43N-(3-Triethylammoniumpropyl) -4-(4-(Dibutylamino) Styryl) PyridiniumDibromide) (5 as per the methodology described earlier.

Flow-Cytometry measurements of the stained bacterial samples wereperformed. In order to adjust the flow-cytometry to the spectralcharacterization as shown in the fluorescence spectra of the FM® 1-43(43N-(3-Triethylammoniumpropyl)-4-(4-(Dibutylamino) Styryl) PyridiniumDibromide) stained bacteria, the [CONFIRM: 488] nm excitation channel ofthe flow-cytometry and two fluorescence channel using 530 (30) nm and610 (20) nm as the λ1 and λ2 for the SIR calculation were used. Theresults are tabulated below and depicted in FIGS. 14A-14C.

TABLE 6 SIR calculation by Flow-Cytometry analysis for E.coli K12 No 700μg 1500 μg antibiotic (A) ampicillin (B) ampicillin (C) LIVE DEAD LIVEDEAD LIVE DEAD % from  94.6%   4.5%  83.7%   15%  78%   20% population530 nm 395  4575 463  4215 566  4011 610 nm 387 16264 440 18753 55921370 SIR 1   3.55  1   4.4  1   5.3

These data demonstrate that the SIR technique is applicable to bacteriakilled or inactivated by antimicrobial drugs.

What is claimed is:
 1. A method for differentiating between live andinactivated bacteria in a treated sample comprising: staining the samplewith a single membrane-associated dye, wherein the singlemembrane-associated dye stains both live and inactivated bacteria;illuminating the sample with an incident light at an excitationwavelength, wherein, when stained with the single membrane-associateddye, an illuminated stained live bacteria exhibits a first maximumemission peak, and wherein an illuminated stained inactivated bacteriaexhibits a second maximum emission peak that is shifted compared to thefirst maximum emission peak; measuring, for each bacterial cell (i) theintensity I1 of emitted light from the single membrane-associated dye atwavelength 1; and (ii) the intensity 12 of emitted light from the singlemembrane-associated dye at wavelength 2, wherein I1 and I2 are from thesame single membrane-associated dye; calculating a ratio I2/I1; anddetermining whether the bacterial cell is live or inactivated based onthe calculated I2/I1 ratio.
 2. The method of claim 1, wherein themembrane-associated dye is N-(3-Triethylammoniumpropyl)-4-(4-(Dibutylamino) Styryl) Pyridinium Dibromide.
 3. The method ofclaim 1, wherein the membrane-associated dye isN-(3-Triethylammoniumpropyl) -4-(6-(4-(Diethylamino) Phenyl)Hexatrienyl) Pyridinium Dibromide.
 4. The method of claim 1, wherein theexcitation wavelength is between 488 and 570 nm.
 5. The method of claim4, wherein the excitation wavelength is 488 nm.
 6. The method of claim1, wherein the emission wavelength range is between 520 and 850 nm. 7.The method of claim 1, wherein wavelength 1 is 530 nm and wavelength 2is 610 nm.
 8. The method of claim 1, wherein wavelength 1 is 670 andwavelength 2 is 780 nm.
 9. The method of claim 1, wherein live bacteriahave a I2/I1<2.0, and inactivated bacteria have a I2/I1>2.0.
 10. Themethod of claim 1, wherein the step of determining whether the bacterialcell is live or inactivated based on the calculated I2/I1 ratiocomprises calculating a second I2/I1 ratio of a control containing onlylive bacteria; and determining whether the bacterial cell is live orinactivated by comparing the I2/I1 ratio of the sample with the secondI2/I1 ratio of the control containing only live bacteria.
 11. The methodof claim 10, wherein the I2/I1 ratio of the sample is divided by thesecond I2/I1 ratio of the control containing only live bacteria togenerate a non-viability parameter, wherein live bacteria have anon-viability parameter≤1.0, and inactivated bacteria have anon-viability parameter>1.0.
 12. A method for differentiating betweenlive and inactivated bacteria in a whole sample comprising the steps of:staining the whole sample with a single membrane-associated dye;illuminating the whole sample with an incident light at an excitationwavelength, measuring, for the stained whole sample, (i) the intensityI1 of emitted light at wavelength 1; and (ii) the intensity I2 ofemitted light at wavelength 2, wherein the I1 and I2 are from the samesingle membrane-associated dye; calculating a ratio I2/I1 of the wholesample and a non-viability parameter (NVP) by dividing the I2/I1 of thewhole sample by I2/I1 of a control containing only live bacteria; andcomparing said NVP to a threshold value to determine if the bacteria arelive or inactivated.
 13. The method of claim 12, wherein the singlemembrane-associated dye is a styryl dye or a cyanine dye.
 14. The methodof claim 12, wherein the excitation wavelength is a wavelength selectedbetween the range of 360 and 570 nm.
 15. The method of claim 12, whereinthe excitation wavelength is a wavelength selected between the range of488 and 570 nm.
 16. The method of claim 12, wherein the range of emittedlight at wavelength 1 lies between 520 and 850 nm.
 17. The method ofclaim 12, wherein the range of emitted light at wavelength 1 liesbetween 530 and 750 nm.
 18. The method of claim 12, wherein the range ofemitted light at wavelength 1 lies between 520 and 700 nm.
 19. Themethod of claim 12, wherein wavelength 1 is 530 nm and wavelength 2 is610 nm.
 20. The method of claim 12, wherein wavelength 1 is 670 andwavelength 2 is 780 nm.
 21. The method of claim 12, further comprisingtreating live bacteria cells contained in the whole sample in an effortto inactivate the bacteria cells prior to the staining step.
 22. Themethod of claim 12, wherein the sample is a treated sample.
 23. Themethod of claim 21, further comprising the step of filtering the stainedcells prior to the illuminating step and subsequent to the stainingstep.
 24. The method of claim 21, wherein the treating step comprisesinteracting the live bacteria with an antimicrobial drug.
 25. The methodof claim 21, wherein the treating step comprises heating, adding anantibiotic drug, adding an antibacterial drug, adding ethanol, orultraviolet (UV) irradiation.
 26. The method of claim 12, wherein thecontrol containing only live bacteria has the same bacterialconcentration as the sample.
 27. The method of claim 12, wherein thethreshold value is 1.0, live bacteria have a non-viabilityparameter≤1.0, and inactivated bacteria have a non-viabilityparameter>1.0.
 28. A method for differentiating between live andinactivated bacteria in a whole sample and stained with a singlemembrane-associated dye, comprising the steps of: illuminating thestained whole sample with an incident light at an excitation wavelength;measuring, for the stained whole sample, (i) the intensity I1 of emittedlight at wavelength 1, and (ii) the intensity I2 of emitted light atwavelength 2, wherein the I1 and I2 are from the same singlemembrane-associated dye; calculating a ratio I2/I1 of the whole sampleand a non-viability parameter (NVP) by dividing the I2/I1 of the wholesample by I2/I1 of a control containing only live bacteria; andcomparing said NVP to a threshold value to determine if the bacteria arelive or inactivated.
 29. The method of claim 28, further comprisingtreating live bacteria cells contained in the sample in an effort toinactivate the bacteria cells prior to the staining step.
 30. The methodof claim 28, wherein the whole sample is a treated sample.
 31. Themethod of claim 28, further comprising the step of filtering the stainedcells prior to the illuminating step and subsequent to the stainingstep.
 32. The method of claim 29, wherein the treating step comprisesinteracting the live bacteria with an antimicrobial drug.
 33. The methodof claim 29, wherein the treating step comprises heating, adding anantibiotic drug, adding an antibacterial drug, adding ethanol, orultraviolet (UV) irradiation.